DNA sequences, vectors, and fusion polypeptides to increase secretion of desired polypeptides from filamentous fungi

ABSTRACT

The invention includes novel fusion DNA sequences encoding fusion polypeptides which when expressed in a filamentous fungus result in the expression of fusion polypeptides which when secreted result in increased levels of secretion of the desired polypeptide as compared to the expression and secretion of such polypeptides from filamentous fungi transformed with previously used DNA sequences. The fusion DNA sequences comprise from the 5&#39; terminus four DNA sequences which encode a fusion polypeptide comprising, from the amino to carbonyl-terminus, first, second, third and fourth amino acid sequences. The first DNA sequence encodes a signal peptide functional as a secretory sequence in a first filamentous fungus. The second DNA sequence encodes a secreted polypeptide or portion thereof which is normally secreted from the same filamentous fungus or a second filamentous fungus. The third DNA sequence encodes a cleavable linker polypeptide while the fourth DNA sequence encodes a desired polypeptide. When the fusion DNA sequence is expressed either in the first or second filamentous fungus, increased secretion of the desired polypeptide is obtained as compared to that which is obtained when the desired polypeptide is expressed from DNA sequences encoding a fusion polypeptide which does not contain the second polypeptide normally secreted from either of the filamentous fungi.

This is a continuation of application Ser. No 08/318,491 filed Oct. 5,1994, now U.S. Pat. No. 5,679,543, which is a continuation ofapplication Ser. No. 08/207,805 filed Mar. 7, 1994, now abandoned, whichis a continuation of application Ser. No. 07/794,603, filed Nov. 15,1991, now abandoned, which is a continuation of application Ser. No.07/369,698, filed Jun. 16, 1989, now abandoned, which is acontinuation-in-part of application Ser. No. 07/163,219, filed Feb. 26,1988, now abandoned, which is a continuation of application Ser. No.06/882,224, filed Jul. 7, 1986, now abandoned, which is acontinuation-in-part of application Ser. No. 06/771,374, filed Aug. 29,1985, now abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to increased secretion of desiredpolypeptides from filamentous fungi. The invention discloses DNAsequences, vectors, fusion polypeptides, and processes for obtainingenhanced production and secretion levels of the desired polypeptide.More particularly, the invention discloses DNA sequences, vectors,fusion polypeptides and processes for the increased secretion of bovinechymosin from filamentous fungi.

BACKGROUND OF THE INVENTION

One of the earlier successes of recombinant DNA technology involved theintracellular expression of the A and B chains of insulin in bacteria ascarboxyl fusions to β-galactosidase. Goeddel, D. V. et al. (1979); Proc.Natl. Acad. Sci. USA 76, 106-110; Johnson, I. S. (1983) Science 219,632-637. Since then, numerous examples have been described for theexpression of fusion polypeptides comprising, in part, a heterologouspolypeptide. Marston, F. A. O. (1986) Biochem. J. 240, 1-12 summarizesthe production of heterologous polypeptides in E.coli. As describedtherein, a number of heterologous polypeptides have been expressedintracellularly as fusion polypeptide in E. coli. In addition,heterologous polypeptides have reportedly been secreted into theperiplasmic space of such microbes by fusing the heterologouspolypeptide with a signal sequence. In some cases, the heterologouspolypeptide was secreted from E. coli into the culture medium whenexpressed with a signal sequence of bacterial origin. In those caseswhere a heterologous protein has been expressed as a fusion with theentire native protein of the host bacteria, the rational was primarilyto increase stability or ease the purification of the fusionpolypeptide.

For example, Scholtissek, S. et al. (1988) Gene 62 55-64, report theexpression in E. coli of a triprotein consisting of bacterialβ-galactosidase, a collagenase recognition site and the single strandedDNA binding protein from E. coli. The β-galactosidase portion of thisfusion polypeptide reportedly was used to purify the fusion polypeptidefrom a crude cell lysate by affinity chromatography on APTG-Sepharose.The single stranded DNA binding protein from E. coli was thereafterisolated from the fusion polypeptide by reacting the collagenaserecognition site with collagenase. Similarly, Smith, D. B. et al. (1988)Gene 67, 31-40 report the bacterial expression of a vector encoding afusion polypeptide consisting of glutathione S-transferase fused at itsC-terminus with a recognition site for blood coagulation factor X_(a)which itself is fused to either of two heterologous polypeptidescorresponding to different antigens of P. falciparum. In anotherexample, Guan, C. et al. (1988) Gene 67, 21-30 report the expression andpurification of fusion polypeptides consisting of maltose bindingprotein fused either to β-galactosidase or PstI Endonuclease, and afusion protein consisting of the bacterial phoA signal, maltose bindingprotein and phoA protein. In the former cases, the fusion polypeptideswere extracted from crude bacterial lysates by affinity chromatographyon cross-linked amylose whereas, in the latter, the fusion protein wasobtained from the periplasmic space after spheroplast formation andaffinity chromatography on cross-linked amylose.

The expression of fusion polypeptides in yeast has also been reported.For example, Cousens, L. S. et al. (1987) Gene 61, 265-275, describe afusion polypeptide consisting of a human superoxide dismutase-humanproinsulin fusion protein with a methionine residue at the junction ofthe two proteins. Superoxide dismutase is an intracellular protein andthe fusion polypeptide was reportedly expressed as an insolubleinclusion body within the yeast expression host with incorrect disulfidebonds. After sulfitollysis proinsulin was reportedly purified, renaturedand processed to yield insulin after cleavage of the methionine residuewith cyanogen bromide.

U.S. Pat. No. 4,751,180 to Cousens et al. states that a polypeptide ofinterest may be obtained in high yield from an expression host, such asyeast, when the polypeptide of interest is expressed as a completelyheterologous fusion polypeptide. One of the heterologous polypeptides isproduced in high yield in the expression host typically in amountsgreater than five percent of the total protein produced by the host. Theonly high yield heterologous polypeptide disclosed, however, is that ofthe intracellular protein human superoxide dismutase which is fused toeither proinsulin or IgF-2. The specification also states that asecretory leader and processing signal may be included as part of thefused polypeptide. No example is provided which indicates that secretionwould be obtained and, if obtained, would be at levels higher than thatwhich have been obtained using a fusion construction which detected thehigh yield heterologous protein in a fusion consisting of a secretoryleader sequence fused to only the polypeptide of interest, e.g.proinsulin or insulin-like growth factor (IgF-2).

Heterologous gene expression has also been reported in filamentousfungi. For example, Christensen, T. et al. (1988) Bio/Technology 6,1419-1422 have reported an expression vector utilizing the α-amylasepromotor from A. oryzae to express the prepro form of aspartylproteinase from the filamentous fungus Rhizomuchor miehei. Whenexpressed in A. oryzae, aspartyl proteinase was obtained from theculture medium. When Gwynne, D. I. et al. (1987) Bio/Technology 5,713-719, report the expression and secretion of human interferon andbacterial endoglucanase from filamentous fungi by expressing these geneswith either a fungal glucoamylase signal or a synthetic consensus signalsequence.

Upshall, A. et al. (1987) Bio/Technology 5, 1301-1304 report theexpression and secretion of human tissue plasminogen activator byexpressing the gene encoding the pre-form of t-PA in a filamentousfungus. Further, Turnbull, I. F. et al. (1989) Bio/Technology 7, 169-174report an attempt to express and secrete bacterial enterotoxin subunit Bfrom filamentous fungi. No secreted material, however, was detected.

Bovine prochymosin has reportedly been expressed in Escherichia coli,the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, and infilamentous fungi by the inventor in Asperaillus species. In E. coliprochymosin, with the first four amino acid residues replaced by anamino-terminal fragment of the trpE gene, has reportedly been producedunder the control of the trp promoter (Nishimori, K. et al. (1984) Gene29, 41-49). The fusion protein accumulated as inclusion bodies in thecytoplasm but after appropriate extraction conditions could be activatedto yield mature chymosin.

Moir et al. (1985) (In: Developments in Industrial Microbiology. Vol.26. Underkofler, L. A. (ed.). Society for Industrial Microbiology,Arlington, Va., U.S.A.) described intracellular production ofprochymosin in S. cerevisiae. The protein was synthesized with varioussegments of phosphoglycerate kinase, triosephosphate isomerase orgalactokinase attached to the amino terminus, allowing increasedproduction compared to direct expression from the same promoters. It wassuggested that the increase in production was due to more efficienttranslation of the mRNA. Moir et al. also reported secretion ofprochymosin from S. cerevisiae, in the form of a fusion with the firstfew residues of invertase or alpha factor. The extracellular prochymosinwas activated at low pH to give mature chymosin despite the additionalamino acids on the prosequence. Similarly, activatable prochymosin wassecreted from the yeast Y. lipolytica with either 14 or 90 residues ofnative alkaline extracellular protease attached to the amino terminus(Franke, A. E. et al. (1988) In: Developments in IndustrialMicrobiology. Vol. 29. Pierce, G. (ed.). Society for IndustrialMicrobiology, Arlington, Va., U.S.A.). In this report, no more thanabout 20% of the amino terminus of the protease was used to generate thefusion polypeptides and no apparent advantage accrued from expression asfusion polypeptides. Active calf chymosin has also been produced in thefilamentous fungus Trichoderma reesei (Harkki, A. et al. (1989)Bio/Technology 7, 596-603. The cellobiohydrolase I gene (cbhI) promoterand terminator regions were employed and four different constructionswere made employing different signal sequences fused to prochymosincDNA. Either the chymosin signal sequence, cbhI signal sequence, ahybrid cbhI/chymosin signal sequence or the cbhI signal sequence plus 20amino acids of mature cbhI were fused to the amino terminus ofprochymosin. Slightly better production was obtained from the latterconstruction although insufficient numbers of transformants wereexamined to confirm this. Secretion was inefficient with approximately66% of the chymosin-derived material remaining within the cell oftransformants regardless of the type of vector construction used.

The glaA gene encodes glucoamylase which is highly expressed in manystrains of Aspergillus niger and Aspergillus awamori. The promoter andsecretion signal sequence of the glaA gene have been used to expressheterologous genes in Aspergilli including bovine chymosin inAspergillus nidulans and A. awamori as previously described by theinventors (Cullen, D. et al. (1987) Bio/Technology 5, 713-719) and EPOPublication No. 0 215 594). In the latter experiments, a variety ofconstructs were made, incorporating prochymosin cDNA, either theglucoamylase or the chymosin secretion signal and, in one case, thefirst 11 codons of mature glucoamylase. Maximum yields of secretedchymosin obtained from A. awamori were below 15 mg/l in 50 ml shakeflask cultures and were obtained using the chymosin signal sequenceencoded by pGRG3. These previous studies indicated that integratedplasmid copy number did not correlate with chymosin yields, abundantpolyadenylated chymosin mRNA was produced, and intracellular levels ofchymosin were high in some transformants regardless of the source ofsecretion signal. It was inferred that transcription was not a limitingfactor in chymosin production but that secretion may have beeninefficient. It was also evident that the addition of a small aminoterminal segment (11 amino acids) of glucoamylase to the propeptide ofprochymosin did not prevent activation to mature chymosin. The amount ofextracellular chymosin obtained with the first eleven codons ofglucoamylase, however, was substantially less than that obtained whenthe glucoamylase signal was used alone.

Accordingly, an object of the invention herein is to provide for theexpression and enhanced secretion of desired polypeptides by and fromfilamentous fungi including fusion DNA sequences, expression vectorscontaining such DNA sequences, transformed filamentous fungi, fusionpolypeptides and processes for expressing and secreting high levels ofsuch desired polypeptides.

It is a further object of the invention to provide for the expressionand enhanced secretion of chymosin from filamentous fungi includingfusion DNA sequences, vectors containing such DNA sequences transformedfilamentous fungi, fusion chymosin polypeptides and processes forexpressing and secreting high levels for chymosin.

The references discussed above are provided solely for their disclosureprior to the filing date of the instant case. Nothing herein is to beconstrued as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or priority basedon earlier filed applications.

SUMMARY OF THE INVENTION

In accordance with the above objects, the invention includes novelfusion DNA sequences encoding fusion polypeptides which when expressedin a filamentous fungus result in the expression of fusion polypeptideswhich when secreted result in increased levels of secretion of thedesired polypeptide as compared to the expression and secretion of suchpolypeptides from filamentous fungi transformed with previously used DNAsequences.

The fusion DNA sequences comprise from the 5' terminus four DNAsequences which encode a fusion polypeptide comprising, from the aminoto carbonyl-terminus, first, second, third and fourth amino acidsequences. The first DNA sequence encodes a signal peptide functional asa secretory sequence in a first filamentous fungus. The second DNAsequence encodes a secreted polypeptide or portion thereof which isnormally secreted from the same filamentous fungus or a secondfilamentous fungus. The third DNA sequence encodes a cleavable linkerpolypeptide while the fourth DNA sequence encodes a desired polypeptide.When the fusion DNA sequence is expressed either in the first or secondfilamentous fungus, increased secretion of the desired polypeptide isobtained as compared to that which is obtained when the desiredpolypeptide is expressed from DNA sequences encoding a fusionpolypeptide which does not contain the second polypeptide normallysecreted from either of the filamentous fungi.

The invention also includes expression vectors containing the abovefusion DNA sequence and filamentous fungi transformed with suchexpression vectors. The invention also includes the fusion polypeptideencoded by such fusion DNA sequences.

Further, the invention includes a process for producing a desiredpolypeptide comprising transforming a host filamentous fungus with theabove described expression vector and culturing the host filamentousfungus to secrete the desired polypeptide into the culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the construction of pBRΔgam-arg.

FIG. 2 depicts the disruption of the glaA gene by replacement of the 5.5kb DNA chromosomal fragment with a 4.5 kb fragment from pBRΔgam-argB.

FIGS. 3A and 3B depict the construction of pGRG(1-4).

FIGS. 4A, 4B, 4C and 4D depict the various cassette inserts used togenerate pGRG1 through pGRG4.

FIGS. 5A and 5B depict the construction of pGAMpR.

FIG. 6 depicts the construction of pUCAMpR1.

FIG. 7 depicts the construction of pSG1.

FIG. 8 depicts Southern blot analysis of DNA from transformants ofstrains GC12 and GCΔGAMpR.

FIG. 9 depicts Northern blot analysis of RNA from strain GC12 andtransformants 12grg1-1a and 12gampr4.

FIG. 10 depicts products of in vitro translation of RNA from strain GC12and transformants 12grg1-1a and 12gampr4.

FIG. 11 depicts Western analysis of chymosin in culture supernatants oftransformants 12grg1-1a and 12gampr4.

DETAILED DESCRIPTION

The inventors have discovered that desired polypeptides can be expressedand secreted at levels higher than that previously obtained by fusingthe desired polypeptide with a polypeptide which is normally secretedfrom a filamentous fungus. Previously, the inventors discovered thatheterologous polypeptides such as bovine chymosin and glucoamylase andcarboxyl (=aspartyl) protease from filamentous fungi could be expressedand secreted from Aspergillus species as described in the parentapplications and EPO Publication No. 0 215 594, each of which areexpressly incorporated herein by reference.

For example, the inventors previously achieved expression and secretionof bovine chymosin from Aspergillus nidulans at levels approaching 1.5micrograms per ml of medium when expressed as a fusion between theglucoamylase signal peptide and the pro-form of chymosin. The vectorencoding this particular construction is designated pGRG1 (see FIGS. 3and 4A). However, when the glucoamylase signal peptide together with theglucoamylase propeptide and the first eleven amino acids of glucoamylasewere fused to prochymosin, secretion levels were substantially reducedto about half of the term obtained in the previous construction, i.e.reduced to approximately 0.75 μg per ml of medium. This vector waspreviously identified as pGRG4 and is identified in FIGS. 3 and 4D. Allof the plasmids pGRG1, pGRG3 and pGRG4 (see FIGS. 3 and 4A, B, C and D)have been transformed into A. awamori. The transformant which producedthe greatest amount of extracellular chymosin was obtained using pGRG3.With improvements to culture medium and conditions the highest level ofsecreted chymosin obtained was below 15 μg/mL as measured by enzymeimmunoassay (which will detect inactive and degraged chymosin inaddition to mature chymosin).

By using the fusion DNA constructions described herein, a dramaticincrease in extracellular chymosin has been obtained. In some caseschymosin levels are approximately 20 fold in excess of that obtainedpreviously. Commensurate with this increase in secretion is thereduction in the amount of chymosin maintained intracellularly in thefilamentous fungi expression host. Well over 50% and, in some cases, asmuch as almost 98% of the chymosin produced previously was maintainedintracellularly (see Table II). When the vectors encoding the DNAsequences of the invention herein were used, however, thirty percent orless, and in some cases, less than 1% of the chymosin expressed wasmaintained intracellularly.

The increased secretion levels of chymosin are the result of expressingchymosin in its pro-form as a fusion polypeptide with a polypeptidenormally secreted by a filamentous fungus. In the preferred embodiments,glucoamylase from A. awamori encoded by the glaA gene, including thesecretory signal sequence of glucoamylase is fused to the amino-terminusof prochymosin. The presence of the glucoamylase signal sequence andmature glucoamylase peptide sequences facilitate the enhanced secretionof the fusion polypeptide into the culture medium. Mature chymosin isthen obtained by acidifying the medium to process the chymosinprosequence to produce active chymosin by removal of the propeptide.

As used herein, a "fusion DNA sequence" comprises from 5' to 3' first,second, third and fourth DNA sequences. The "first DNA sequence" encodesa signal peptide functional as a secretory sequence in a firstfilamentous fungus. Such signal sequences include those fromglucoamylase, α-amylase and aspartyl proteases from Aspergillus awamori,Aspergillus niger, Aspergillus oryzae, signal sequences fromcellobiohydrolase I, cellobiohydrolase II, endoglucanase I,endoglucanase III from Trichoderma, signal sequences from glucoamylasefrom Neurospora and Humicola as well as signal sequences from eukaryotesincluding the signal sequence from bovine chymosin, human tissueplasminogen activator, human interferon and synthetic consensuseukaryotic signal sequences such as that described by Gwynne (1987)supra. Particularly preferred signal sequences are those derived frompolypeptides secreted by the expression host used to express and secretethe fusion polypeptide. For example, the signal sequence fromglucoamylase from Asperaillus awamori is preferred when expressing andsecreting a fusion polypeptide from Aspergillus awamori. As used herein,first amino acid sequences correspond to secretory sequences which arefunctional in a filamentous fungus. Such amino acid sequences areencoded by first DNA sequences as defined.

As used herein, "second DNA sequences" encode "secreted polypeptides"normally expressed from filamentous fungi. Such secreted polypeptidesinclude glucoamylase, α-amylase and aspartyl proteases from Aspergillusawamori, Aspergillus niger, and Aspergillus oryzae, cellobiohydrolase I,cellobiohydrolase II, endoglucanase I and endoglucanase III fromTrichoderma and glucoamylase from Neurospora species and Humicolaspecies. As with the first DNA sequences, preferred secretedpolypeptides are those which are naturally secreted by the filamentousfungal expression host. Thus, for example when using Aspergillusawamori, preferred secreted polypeptides are glucoamylase and α-amylasefrom Aspergillus awamori, most preferably glucoamylase.

As used herein, "third DNA sequences" comprise DNA sequences encoding acleavable linker polypeptide. Such sequences include those which encodethe prosequence of bovine chymosin, the prosequence of subtilisin,prosequences of retrovirul proteases including human immunodeficiencyvirus protease and DNA sequences encoding amino acid sequencesrecognized and cleaved by trypsin, factor X_(a) collagenase, clostripin,subtilisin, chymosin, yeast KEX2 protease and the like. See e.g.Marston, F. A. O. (1986) Biol. Chem J. 240, 1-12. Such third DNAsequences may also encode the amino acid methionine which may beselectively cleaved by cyanogen bromide. It should be understood thatthe third DNA sequence need only encode that amino acid sequence whichis necessary to be recognized by a particular enzyme or chemical agentto bring about cleavage of the fusion polypeptide. Thus, the entireprosequence of, for example, chymosin or subtilisin need not be used.Rather, only that portion of the prosequence which is necessary forrecognition and cleavage by the appropriate enzyme is required.

As used herein, "fourth DNA sequences" encode "desired polypeptides."Such desired polypeptides include mammalian enzymes such as bovinechymosin, human tissue plasminogen activator etc., mammalian hormonessuch as human growth hormone, human interferon, human interleukin andmammalian proteins such as human serum albumin. Desired polypeptidesalso induce bacterial enzymes such as α-amylase from Bacillus species,lipase from Pseudomonas species, etc. Desired polypeptides furtherinclude fungal enzymes such as lignin peroxidase and Mn²⁺ -dependentperoxidase from Phanerochaete, glucoamylase from Humicola species andaspartyl proteases from Mucor species.

The above-defined four DNA sequences encoding the corresponding fouramino acid sequences are combined to form a "fusion DNA sequence." Suchfusion DNA sequences are assembled in proper reading frame from the 5'terminus to 3' terminus in the order of first, second, third and fourthDNA sequences. As so assembled, the DNA sequence will encode a "fusionpolypeptide" encoding from its amino-terminus a signal peptidefunctional as a secretory sequence in a filamentous fungus, a secretedpolypeptide or portion thereof normally secreted from a filamentousfungus, a cleavable linker polypeptide and a desired polypeptide.

As indicated, the first DNA sequence encodes a signal peptide functionalas a secretory signal in a first filamentous fungus. The signalsequences may be derived from a secreted polypeptide from a particularspecies of filamentous fungus. As also indicated, the second DNAsequence encodes a second amino acid sequence corresponding to all orpart of a polypeptide normally secreted by either the first filamentousfungus (from which the signal peptide is obtained) or a secondfilamentous fungus (if the signal peptide and secreted polypeptide arefrom different filamentous fungi or if the signal peptide is obtainedfrom a source other than a filamentous fungus, e.g. the chymosin signalfrom bovine species).

As indicated, all or part of the mature sequence of the secretedpolypeptide is used in the construction of the fusion DNA sequences. Itis preferred that full length secreted polypeptides be used to practicethe invention. However, functional portions of the secreted polypeptidemay be employed. As used herein a "portion" of a secreted polypeptide isdefined functionally as that portion of a secreted polypeptide whichwhen combined with the other components of the fusion polypeptidedefined herein results in increased secretion of the desired polypeptideas compared to the level of desired polypeptide secreted when anexpression vector is used which does not utilize the secretedpolypeptide. Thus, the secretion level of a fusion DNA sequence encodingfirst, second, third and fourth amino acid sequences (the second DNAsequence containing all or a portion of a secreted polypeptide) iscompared to the secretion level for a second fusion polypeptidecontaining only first, third and fourth amino acid sequences (i.e.,without a secreted polypeptide or a portion thereof). Those amino acidsequences from the secreted polypeptide, and DNA sequences encoding suchamino acids, which are capable of producing increased secretion ascompared to the second fusion polypeptide comprise the "portion" of thesecreted polypeptide as defined herein.

Generally, such portions of the secreted polypeptide comprise greaterthan 50% of the secreted polypeptide, preferably greater than 75%, mostpreferably greater than 90% of the secreted polypeptide. Such portionscomprise preferably the amino-terminal portion of the secretedpolypeptide.

The "filamentous fungi" of the present invention are eukaryoticmicroorganisms and include all filamentous forms of the subdivisionEumycotina, Alexopoulos, C. J. (1962), Introductory Mycology, New York:Wiley. These fungi are characterized by a vegetative mycelium with acell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi of the present invention aremorphologically, physiologically, and genetically distinct from yeasts.Vegetative growth by filamentous fungi is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as S. cerevisiae is by budding of a unicellular thallus,and carbon catabolism may be fermentative. S. cerevisiae has aprominent, very stable diploid phase whereas, diploids exist onlybriefly prior to meiosis in filamentous fungi like Aspergilli andNeurospora. S. cervisiae has 17 chromosomes as opposed to 8 and 7 for A.nidulans and N. crassa respectively. Recent illustrations of differencesbetween S. cerevisiae and filamentous fungi include the inability of S.cerevisiae to process Aspergillus and Trichoderma introns and theinability to recognize many transcriptional regulators of filamentousfungi (Innis, M. A. et al. (1985) Science, 228, 21-26).

Various species of filamentous fungi may be used as expression hostsincluding the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Endothia, Mucor,Cochliobolus and Pyricularia. Specific expression hosts include A.nidulans, (Yelton, M., et al. (1984) Proc. Natl. Acad. Sci. USA , 81,1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45;John, M. A. and J. F. Peberdy (1984) Enzyme Microb. Technol. 6, 386-389;Tilburn, et al. (1982) Gene 26, 205-221; Ballance, D. J. et al., (1983)Biochem. Biophys. Res. Comm. 112, 284-289; Johnston, I. L. et al. (1985)EMBO J. 4, 1307-1311) A. niger, (Kelly, J. M. and M. Hynes (1985) EMBO4, 475-479) A. awamori, e.g., NRRL 3112, ATCC 22342, ATCC 44733, ATCC14331 and strain UVK 143f, A. oryzae, e.g., ATCC 11490, N. crassa (Case,M. E. et al. (1979) Proc. Natl. Acad. Scie. USA 76, 5259-5263; LambowitzU.S. Pat. No. 4,486,553; Kinsey, J. A. and J. A. Rambosek (1984)Molecular and Cellular Biology 4, 117-122; Bull, J. H. and J. C. Wooton(1984) Nature 310, 701-704), Trichoderma reesei, e.g. NRRL 15709, ATCC13631, 56764, 56765, 56466, 56767, and Trichoderma viride, e.g., ATCC32098 and 32086. A preferred expression host is A. awamori in which thegene encoding the major secreted aspartyl protease has been deleted. Theproduction of this preferred expression host is described in U.S. patentapplication Ser. No. 214,237 filed Jul. 1, 1988, now abandoned,expressly incorporated herein by reference.

As used herein, a "promotor sequence" is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.It is operably linked to a DNA sequence encoding the above definedfusion polypeptide. Such linkage comprises positioning of the promoterwith respect to the translation initiation codon of the DNA sequenceencoding the fusion DNA sequence. The promoter sequence containstranscription and translation control sequences which mediate theexpression of the fusion DNA sequence. Examples include the promoterfrom the A. awamori or A. niger glucoamylase genes (Nunberg, J. H. etal. (1984) Mol. Cell. Biol. 4, 2306-2315; Boel, E. et al. (1984) EMBO J.3, 1581-1585), the Mucor miehei carboxyl protease gene herein, theTrichoderma reesei cellobiohydrolase I gene (Shoemaker, S. P. et al.(1984) European Patent Application No. EP00137280A1), the A. nidulanstrpC gene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81,1470-1474; Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45)the A. nidulans alcA gene (Lockington, R. A. et al. (1986) Gene 33,137-149), the A. nidulans tpiA gene (McKnight, G. L. et al. (1986) Cell46, 143-147), the A. nidulans amdS gene (Hynes, M. J. et al. (1983) Mol.Cell Biol. 3, 1430-1439), and higher eukaryotic promoters such as theSV40 early promoter (Barclay, S. L. and E. Meller (1983) Molecular andCellular Biology 3, 2117-2130).

Likewise a "terminator sequence" is a DNA sequence which is recognizedby the expression host to terminate transcription. It is operably linkedto the 3' end of the fusion DNA encoding the fusion polypeptide to beexpressed. Examples include the terminator from the A. nidulans trpCgene (Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474;Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45), the A.awamori or A. niger glucoamylase genes (Nunberg, J. H. et al. (1984)Mol. Cell. Biol. 4, 2306-253; Boel, E. et al. (1984) EMBO J. 3,1581-1585), and the Mucor miehei carboxyl protease gene (EPO PublicationNo. 0 215 594), although any fungal terminator is likely to befunctional in the present invention.

A "polyadenylation sequence" is a DNA sequence which when transcribed isrecognized by the expression host to add polyadenosine residues totranscribed mRNA. It is operably linked to the 3' end of the fusion DNAencoding the fusion polypeptide to be expressed. Examples includepolyadenylation sequences from the A. nidulans trpc gene (Yelton, M. etal. (1984) Proc. Natl. Acad. Sci. USA 81, 1470-1474; Mullaney, E. J. etal. (1985) Mol. Gen. Genet. 199, 37-45), the A. awamori or A. nigerglucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell. Biol. 4,2306-2315) (Boel, E. et al. (1984) EMBO J. 3, 1581-1585), and the Mucormiehei carboxyl protease gene described above. Any fungalpolyadenylation sequence, however, is likely to be functional in thepresent invention.

Materials and Methods

General methods were as previously described in EPO Publication 0 215594.

Strains

The Aspergillus awamori strains used in this work were all derived froma glucoamylase over-producing strain (UVK143f), itself derived fromNRRL3112 as described in EPO Publication No. 0 215 594. Strain genotypeswere: strain GC12 (pyrG5; argB3) (derived from strain pyr4-5, also knownas GC5, as described in U.S. patent application Ser. No. 214,237) andstrain GCΔGAM23 (pyrG5; ΔglaA23).

Strain GCΔGAM23 was derived from strain GC12 by disruption of theglucoamylase (glaA) gene. This was achieved by transformation with alinear DNA fragment (similar to the method described by Miller et al.(1985) Mol. Cell. Biol. 5, 1714-1721) having glaA flanking sequences ateither end and with 2.7 kb of the promoter and coding region of the glaAgene replaced by the Aspergillus nidulans argB gene as selectablemarker. The vector from which we obtained this linear fragment of DNAwas assembled as follows (FIG. 1). A 5.5 kb ClaI fragment of DNAcontaining approximately 3.5 kb of 5' flanking DNA and approximately 2kb of coding sequence of the A. awamori UVK143f glaA gene was clonedinto the ClaI site of pBR322. This plasmid was cut with restrictionendonucleases XhoI and BglII to remove a section of DNA extending from aposition 1966 bp upstream from the translation start codon to a positionfollowing approximately 200 codons of coding sequence. The overhangingDNA ends were filled in using the Klenow fragment of DNA polymerase Iand ligated to reconstitute a BglII cleavage site and give pBRΔGAMXB. A1.7 kb BamHI fragment containing the Aspergillus nidulans argB gene wascloned into this reconstituted BglII site to create the vectorpBRΔgam-argB4 shown in FIG. 1. This vector was cut with ClaI and used totransform strain GC12 using complementation of the argB mutation toselect for transformants. Integration of the linear fragment containingthe glaA flanking sequences and the argB gene at the chromosomal glaAlocus was identified by Southern blot analysis. Briefly, DNA fromtransformants and strain GC12 was digested with ClaI, subjected toagarose gel electrophoresis, transferred to a membrane filter andhybridized with a radiolabelled fragment of DNA containing the A. nigerglaA gene. Two bands (5.5 and 1.9 kb in size) were observed inuntransformed GC12 DNA following autoradiography representing thechromosomal glaA gene (data not shown). The predicted alteration due todisruption of the glaA gene was replacement of the 5.5 kb DNA fragmentwith a fragment of 4.5 kb (FIG. 2). This change had occurred intransformed strain GCΔGAM23. Enzyme immunoassays specific forglucoamylase confirmed that this strain did not secrete detectablelevels of glucoamylase.

Media

Aspergillus complete and minimal media (Rowlands, R. T. et al. (1973)Mol. Gen. Genet. 126, 201-216) were used for the growth of fungalcolonies and were supplemented with 2 mg/ml arginine or uridine asrequired. Two different liquid media were used to study chymosinproduction in shake flasks. SCM consisted of maltose, 50 g/l; maltextract, 20 g/l; yeast extract, 5 g/l; bacto-peptone, 1 g/l; arginine, 1g/l; uridine, 1 g/l; methionine, 0.5 g/l; biotin, 2 mg/l; streptomycin,50 mg/l; KH₂ PO₄, 34 g/l; NaNO₃, 6 g/l; MgSO₄.7H₂ O, 1 g/l; KCl, 0.52g/l; trace elements solution (18), 1 ml/l; Tween-80, 1 ml/l; Mazu DF60-Pantifoam (Mazur chemicals Inc.), 2 ml/l; pH5. Soy medium containedmaltose, 150 g/l; soy bean meal or soluble soy-milk powder, 60 g/l;sodium citrate, 70 g/l; (NH₄)₂ SO₄, 15 g/l; NaH₂ PO₄, 1 g/l; MgSO₄, 1g/l; Tween-80, 1 ml/l; Mazu DF60-P antifoam 2 ml/l; arginine 1 g/l;uridine, 1 g/l; streptomycin 50 mg/l; pH 6.2.

Fungal Transformation

Polyethylene glycol (PEG) mediated transformation was performed asdescribed previously Cullen, D. et al. (1987) Bio/Technology 5, 369-376except that 0.7 M KCl was used during preparation of protoplasts and wasalso added to the PEG solution. In addition, aurintricarboxylic acid (10μg/ml) was added to the final protoplast wash prior to PEG treatment. Wehave observed that this nuclease inhibitor increased transformationfrequencies for A. awamori by 2-5 fold and had little effect onprotoplast viability (data not shown).

Transformation by electroporation was performed as described by Ward etal (1988) Curr. Genet. 14, 37-42. Briefly, washed protoplasts weresuspended in electroporation buffer (7 mM sodium phosphate, pH 7.2, 1 mMMgSO₄, 1.4 M sorbitol), DNA was added and a pulse of 2,125 V/cm wasdelivered from the 25 μFD capacitor of a Bio-Rad Gene Pulser apparatus.

Following either method of transformation protoplasts were plated ontosolidified Aspergillus minimal medium with 1.2 M sorbitol and lackinguridine.

DNA and RNA Manipulation

Standard methods were used for plasmid isolation, restriction enzymedigestion, ligation of DNA, DNA fragment isolation, DNAdephosphorylation, nick translation and Southern analysis (Maniatis, T.et al (1982) Molecular Cloning. A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Fungal DNA was isolated aspreviously described (Cullen, D. et al (1982) Bio/Technology 5,369-376).

Total RNA was extracted from fungi (24) and poly(A)⁺ RNA was selected onoligo(dT) columns by standard procedures Maniatis (1982) supra. RNA waselectrophoresed in formaldehyde-agarose gels prior to blotting tomembrane filters for Northern analysis (Id.).

In vitro Translation

In vitro translation of poly(A)⁺ RNA from A. awamori was done usingrabbit reticulocyte lysates (Bethesda Research Laboratories,Gaithersberg, Md.). Each 60 μl reaction contained the following: 2.6 μl2M potassium acetate, pH 7.2, 3 μl 20 mM magnesium acetate, pH 7.2, 10μl (100 μCi) ³⁵ S-cysteine (Amersham, Arlington Heights, Ill.), 20 μlreaction buffer (Bethesda Research Laboratories, cat. no. 8112), 40 μlrabbit reticulocyte lysate (Bethesda Research Laboratories, cat. no.8111), 36.8 μl of water and RNA (approximately 10 μg). The reactionswere incubated at 30° C. for 60 minutes, then stopped by placing on ice.Incorporation of the ³⁵ S-cysteine was measured by precipitation withcold trichloroacetic acid (10% v/v).

Immunoprecipitation of radiolabelled chymosin polypeptides was done bythe following method: 50 μl of ³⁵ S-labelled in vitro translationreaction was mixed with an equal volume of 2× NETS buffer (1× NETSbuffer is 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.4, 0.05% TritonX-100 and 0.25% gelatin). In order to remove proteins whichnon-specifically bind to protein A, 20 μl Pansorbin (protein A bearingStaphylococcus aureus cells; Calbiochem, La Jolla, Calif.) was added,mixed and incubated for 30 minutes at room temperature. The Pansorbincells had previously been washed twice in 1× NETS and resuspended intheir original volume (10% suspension). After incubating, the mixturewas centrifuged and the supernatant was placed in a clean tube. Next 30μl of chymosin antibody (purified by affinity chromatography andadjusted to a final concentration of 430 μg/ml in 1× NETS) was added andthe mixture was incubated for 2 hours at room temperature. Followingincubation, 50 μl of washed Pansorbin cells were added. The suspensionwas mixed thoroughly and incubated for 1 hour at room temperature.Subsequently, the mixture was centrifuged and the pellet was washedthree times in 1× NETS. Lastly, the pellet was resuspended in 25 μl ofwater, mixed with an equal volume of sample buffer (1% SDS, 25 mMglycine, 192 mM Tris, pH 8.3, 50% sucrose, 50 mM β-mercaptoethanol) andheated to 95° C. for five minutes prior to SDS-polyacrylamide gelelectrophoresis.

Chymosin Production by Transformants

50 ml of SCM or soy medium in 250 ml shake flasks were inoculated withfresh spore suspensions and cultured at 37° C. Samples were assayed forchymosin protein by enzyme immunoassay "EIA" Engvall, E. (1980) MethodsEnzymol 70, 419-439 using rabbit anti-chymosin antibody and authenticcalf chymosin (Chris Hansens Laboratorium, Denmark) as standard.

Chymosin activity assays were performed in microtitre plates and werebased on an increase in turbidity due to milk clotting. 25 μl of samplewas diluted in 10 mM sodium phosphate, pH 6.0 and 150 μl of substrate(1% skim milk, 40 mM CaCl₂ and 50 mM sodium acetate, pH 6.0) was added.After incubation at 37° C. for 15 min the turbidity was read at 690 nm.Authentic calf chymosin was used as the standard.

To determine intracellular chymosin concentrations mycelium washarvested from 50 ml cultures, washed thoroughly with water, freezedried and ground in a mortar and pestle with sand. 50 ml of extractionbuffer (50 mM sodium phosphate pH 5.5, 0.5 M NaCl, 1 mM phenyl methylsulfonyl fluoride, 0.1 mM pepstatin) was added and mixed thoroughly.Samples of the extract were adjusted to 50 mM NaOH by the addition of 1M NaOH, incubated at 37° C. for 30 min and finally centrifuged(13,000×g) to remove the cell debris. Chymosin concentration in thesupernatant was measured by EIA.

For Western analysis samples were electrophoresed in SDS-polyacrylamidegels and blotted to membrane filters by standard procedures (Towbin, H.et al (1979) Proc. Natl. Accad. Sci. u.s.a. 76, 4350-4354). Blots weresequentially treated with rabbit anti-chymosin and goat anti-rabbit IgGconjugated with horse radish peroxidase (HRP). HRP color development wasthen performed by incubation with H₂ O₂ and 4-chloro-1-napthol.

EXAMPLE 1 Increased Chymosin Secretion from A. awamori

Construction of pGAMpR

Construction of the chymosin expression vectors pGRG1 and pGRG3 has beendescribed previously in Cullen, D. et al. (1987) Bio/Technology 5,369-376 and EPO Publication No. 0 215 594. They consist of an expressioncassette comprising an A. niger glaA promoter and terminator, either theglucoamylase or chymosin secretion signal, and the prochymosin B cDNAcoding sequence. This cassette is present in pDJB3 (Ballance, D. J. etal. (1985) Gene 36, 321-331) which consists of pBR325, the N. crassapyr4 gene and the ans1 sequence isolated from A. nidulans and conferringhigh transformation frequency in A. nidulans (FIG. 3). See also FIGS. 4Athrough 4D which depict the cassette inserts used to produce pGRG1through pGRG4 respectively.

The vector pGAMpR contained prochymosin B cDNA sequences fused in frameto the last codon of the A. awamori glaA gene. Construction of thisvector is outlined in FIGS. 5A and 5B. Briefly, a syntheticoligonucleotide (a 54 bp SalI-BamHI fragment encoding the last tencodons of glucoamylase and the first six codons of prochymosin) wascloned in an M13 vector and its nucleotide sequence verified. Into thesame M13 vector we inserted a 235 bp BAMHI-Asp718 fragment from pR1(Cullen (1987) supra and EPO Publication No. 0 215 594) comprising the5' portion of the prochymosin coding sequence beginning at the seventhcodon. From the resulting vector, designated M13mp19GAM3'-5'PR, a 280 bpSalI-Asp718 fragment was isolated and used in a three-part ligation witha 2.3 kb SalI-MluI fragment containing most of the A. awamoriglucoamylase coding region plus 0.5 kb of 5' flanking DNA and anMluI-Asp718 vector fragment containing a pBR322 replicon, the 3' portionof prochymosin, the glucoamylase terminator region from A. niger, and a1.4 kb segment (XhoI-MluI) of the A. awamori glucoamylase promoterregion. The plasmid produced from this ligation, pBR-GAMpR, was digestedwith ClaI and ligated with a 2.1 kb ClaI fragment encoding the pyr4 genefrom Neurospora crassa (Buxton, F. P. et al. (1983) Mol. Gen. Genet.190, 403-405) to derive the final vector pGAMpR (FIGS. 5A and 5B).

Chymosin Production Levels

Protoplasts of strain GC12 and GCΔGAM23 were transformed, using PEG orelectroporation, with plasmids pGRG1, pGRG3, and pGAMpR (FIGS. 3, 4A,4C, 5A and 5B). These plasmids all included the N. crassa pyr4 genewhich is capable of complementing the pyrG mutation of A. awamori, soallowing selection of transformants. Transformants with designationsbeginning with the number 12 are in strain GC12, those beginning with 23are in GCΔGAM23. The name of the plasmid used for transformation isincluded in the designation. Following purification spores from thetransformants were inoculated into 50 ml of SCM and cultured for 4 days.Replicate cultures of individual transformants were not performed and noattempt was made to correct for different growth rates. Immunoassayswere performed on the culture supernatants and on intracellular extractsof the mycelium (Table 1). Treatment of the intracellular extract withNaOH was required to release chymosin from the insoluble cellulardebris. However, this treatment was also found to decrease the amount ofdetectable chymosin in standard samples using EIA by approximately 25%.Thus, the values recorded in Table 1 for intracellular chymosin areunderestimates.

                  TABLE I                                                         ______________________________________                                        Table 1. Concentration of chymosin in samples                                 from 4 day old SCM cultures                                                            Chymosin Concentration (μg/ml)                                              Intra-        Extra-     % Intra-                                   Strain    cellular      cellular   cellular                                   ______________________________________                                        GC12      N.D.          N.D.                                                  GCΔGAM23                                                                          N.D.          N.D.                                                  12grg1-1  5.4           1.2        81.8                                       12grg1-1a 20.8          0.5        97.7                                       12grg1-3a 0.6           1.1        35.3                                       12grg1-4a 2.5           1.4        64.1                                       12grg1-5a 4.6           0.8        85.2                                       12grg3-3a 2.5           3.7        40.3                                       12grg3-5a 6.0           0.9        87.0                                       12grg3-6a 1.7           1.2        58.6                                       12grg3-7a 1.5           1.3        53.6                                       12grg3-9a 0.8           3.2        20.0                                       23grg1-1a 4.4           1.4        75.9                                       23grg1-2a 20.1          0.6        97.1                                       23grg1-3a 4.1           0.7        85.4                                       23grg1-5a 5.4           0.8        87.1                                       23grg3-1a 9.2           0.1        98.9                                       23grg3-2a 8.1           0.4        95.3                                       23grg3-3a 20.8          0.2        99.0                                       23grg3-6a 2.7           0.6        81.8                                       23grg3-7a N.D.          0.1        0.0                                        12gampr1  0.3           0.7        30.0                                       12gampr2  0.5           4.3        10.4                                       12gampr3  N.D.          1.2        0.0                                        12gampr4  2.0           33.6       5.6                                        12gampr31.sup.a                                                                         0.6           26.6       2.2                                        12gampr58.sup.a                                                                         0.8           24.9       3.1                                        23gampr1  0.3           2.0        13.0                                       23gampr3  N.D.          0.2        0.0                                        23gampr4  0.6           47.5       1.2                                        23gampr5  0.3           41.5       0.7                                        23gampr6  0.1           19.7       0.5                                        23gampr7  0.8           42.7       1.8                                        ______________________________________                                         N.D.: not detected;                                                           .sup.a transformants selected as high producers.                         

None of the pGRG1 or pGRG3 transformants, expected to synthesizepreprochymosin without fusion to glucoamylase, gave levels of secretedchymosin greater than 3.7 μg/ml. As noted previously using strain GC5(see U.S. patent application Ser. No. 214,237, abandoned) many of thepGRG1 and pGRG3 transformants had high intracellular levels of chymosin,with greater than 75% of the total chymosin produced remaining withinthe cell in many of the transformants. In contrast, several of thepGAMpR transformants secreted comparatively high levels of chymosin andin the majority of cases the intracellular levels of chymosin were muchlower than the amounts of secreted chymosin. At this time it was notedthat higher expression levels could be obtained in soy medium, possiblyrelated to the higher pH of this medium which might reduce the activityof native, secreted aspartyl proteases. Strains 12grg1-1a, 12gampr4 and23gampr46 (not shown in Table 1) were chosen as the highest producersfor further study. The levels of intracellular and extracellularchymosin produced by triplicate, 6 day old, 50 ml Soy bean meal mediumcultures of these strains were measured by EIA and activity assays. Inaddition, glucoamylase concentrations in the culture supernatants weremeasured by EIA (Table II).

                  TABLE II                                                        ______________________________________                                        Table 2 Chymosin and glucoamylase production by                               transformants expressed as milligrams per gram dry                            weight of mycelium.                                                                              Extra-           Intra-                                                       cellular   Glyco-                                                                              cellular                                            Chymosin Chymosin   amylase                                                                             Chymosin                                  Transformant                                                                            Activity EIA        EIA   EIA                                       ______________________________________                                        12grg1-1a 1.0      1.3        64.1  0.6                                       12gampr4  22.0     27.7       146.0 0.0                                       23gampr46 14.3     21.7       59.1  0.7                                       ______________________________________                                    

In all cases the amount of secreted chymosin detected by EIA was greaterthan that detected by activity assays. This may reflect the presence ofinactive or degraded chymosin molecules. The results confirmed the highlevels of secreted chymosin produced by transformants expressingchymosin as a fusion protein. Approximately 140 μg of active chymosinwas secreted per ml of culture by transformant 12gampr4 compared toapproximately 8 μg/ml for 12grg1-1a.

The only glucoamylase produced by transformant 23gampr46 (deleted forthe native glaA gene) would be as part of the glucoamylase-chymosinfusion protein. Since the sizes of the two forms of glucoamylase (MW61,000 and 71,000) are approximately twice that of chymosin (MW 37,000)one would expect double the amount, by weight, of glucoamylase to besecreted compared to chymosin. In fact, the measured ratio ofglucoamylase to chymosin in the culture medium was closer to 3:1. Thisdiscrepancy may be due to inaccuracies in the assays or may indicatethat degradation of chymosin has occurred. In transformant 12grg1-1aonly native glucoamylase would be produced whereas in 12gampr4 bothnative and chymosin-associated glucoamylase would be secreted.Interestingly, almost as much recombinant glucoamylase was produced in23gampr46 as native glucoamylase in 12grg1-1a. Although a highpercentage (32%) of the total amount of chymosin produced by 12grg1-1aremained within the cell this was not nearly as dramatic as theintracellular accumulation observed in SCM culture.

Southern Blot Analysis

DNA was extracted from strains GC12 and GCΔGAM23 and from transformants12gampr2, 12gampr3, 12gampr4, 23gampr1 and 23gampr46, digested with XhoIand HindIII, and subjected to electrophoresis. After blotting ontomembrane filters the DNA was hybridized with radiolabelled pGAMpR (FIG.8). For strain GC12 a single band representing the native glaA gene wasobserved (FIG. 8, Lane a). A smaller sized glaA fragment was seen instrain GCΔGAM23 due to the gene replacement event at this locus (FIG. 8,Lane e). The plasmid pGAMpR was also run on the gel to show the size offragments obtained from this on digestion with XhoI and HindIII (FIG. 8,Lane h). Additional bands derived from pGAMpR were observed in thetransformants. For 12gampr4 and 23gampr1 the pattern was consistent withthe integration of a few tandem copies of pGAMpR at a single site awayfrom the glaA locus (FIG. 8, lanes d and f). The number of plasmidcopies in these transformants appears to be similar despite the largedifferences in chymosin productivity. Although tandem plasmidintegration has probably occurred more extensive plasmid rearrangementswere also involved in transformants 12gampr2, 12gampr3 and 23gampr46(FIG. 8, lanes b, c and g).

Northern Analysis

Total RNA was extracted from strains GC12, 12grg1-1a and 12gampr4subjected to electrophoresis and blotted to membrane filters. The RNAwas then hybridized simultaneously with two radiolabelled DNA probes(FIG. 9). One of these probes was a 5 kb EcoR1 fragment containing theA. niger olic gene (Ward et al (1988) Curr. Genet. 14, 37-42) to act asan internal control demonstrating that equivalent amounts of thedifferent RNA samples were applied to the gel and that none of thesamples was excessively degraded. The second probe was an approximately850 bp KpnI-BclI fragment of chymosin coding sequence. In addition tothe oliC mRNA band of approximately 1 kb a 1.4 kb band representingchymosin mRNA was observed in transformant 12grg1-1a (FIG. 9, lane b).It was apparent that abundant chymosin-specific message was present inthis transformant although the level of chymosin production was verymuch lower than glucoamylase production (this strain is capable ofsecreting approximately 0.8 gm/l of glucoamylase). A mRNA species of thesize expected for a fused glucoamylase-chymosin message (3.4 kb) wasobserved in strain 12gampr4 (FIG. 9, lane c). This fused mRNA speciesappeared to be less abundant than the chymosin-specific mRNA present intransformant 12grg1-1a even though chymosin production was much greaterin transformant 12gampr4. Only the olic mRNA was observed in strain GC12(FIG. 9, lane a).

In vitro Translation

Polyadenylated RNA samples isolated from cultures of transformants12gampr4, 23gamp46 and 12grg1-1a were translated in a commercial rabbitreticulocyte in vitro translation system. Chymosin wasimmunoprecipitated from the translation products, subjected toSDS-polyacrylamide gel electrophoresis and visualized by autoradiography(FIG. 10). Two distinct bands, representing proteins of MW 37,000 and42,000, were observed with mRNA from 12grg1-1a (FIG. 10, lane b) whichwas expected to produce only preprochymosin (MW 42,000). The lower MWspecies may represent mature chymosin although autocatalytic processingwould not be expected to take place at the pH at which the translationreactions were performed. With 12gampr4 (FIG. 10, lane a) and 23gampr46mRNA samples two high MW species were precipitated with theanti-chymosin antibody. These were of the approximate size expected forfull-length fusion proteins (100,000 and 110,000 MW) containingprochymosin and either one or the other of the two forms ofglucoamylase. No chymosin could be immunoprecipitated if GC12 RNA (FIG.10, lane c) or no RNA (FIG. 10, lane d) was added to the in vitrotranslation system.

Western Analysis

Supernatants were collected from 50 ml SCM or soy medium cultures oftransformants 12gampr4, 23gampr46 and 12grg1-1a at various time pointsafter inoculation. Samples were separated by SDS-polyacrylamide gelelectrophoresis, blotted to membrane filters and probed with antibodyspecific for chymosin (FIG. 11). No chymosin was observed in the culturesupernatant from strain GC12 (FIG. 11, lane b). Authentic bovinechymosin was also run on the gel (FIG. 11, lanes a and i). A band of thesame size as authentic bovine chymosin (37,000 MW) was observed in allthe samples from SCM cultures of 2 days and older. In soy mediumcultures of 12gampr4 (FIG. 11, lane g) and 23gampr46 an additional bandof approximately the size expected (100,000 MW) for a full-lengthglucoamylase-chymosin fusion protein was evident at 2 and 3 daysalthough this was diminished at later time points. The majorchymosin-specific band present in samples from 12grg1-1a cultures in soymedium at 2 days was of the size predicted for prochymosin (FIG. 11,lane d). Soy medum was buffered at pH 6.2, whereas SCM medium was at pH5. At the higher pH activation of prochymosin would be expected to beslow.

The pH of samples from day 2 or 3 soy medium cultures was lowered to pH2 for 30 min. at room temperature. The pH was then raised immediately toabove pH 6 before loading the sample onto an SDS polyacrylamide gel forWestern analysis. This treatment led to a loss of the large molecularweight band from 12gampr4 (FIG. 11, lane h) or 23gampr46 or loss ofprochymosin from 12grg1-1a (FIG. 11, lane e) and the accumulation of aprotein species slightly larger than mature chymosin, possiblypseudochymosin in all transformants. These changes in the size ofchymosin-specific bands were inhibited if the aspartyl proteaseinhibitor pepstatin (Marciniszyn, J. Jr. et al. (1976) J. Biol. Chem.251, 7095-7102) was included at 0.1 mM during the low pH treatment.Chymosin concentration measured by activity assays on samples from 2 dayold soy medium cultures of 12grg1-1a and 23gampr46 were 0.7 and 3.2μg/ml respectively before treatment at pH 2. Following treatment thesevalues rose to 3.6 and 17.5 μg/ml respectively, an increase ofapproximately 5 fold in each case.

As can be seen from Tables I and II, the yields of secreted chymosin inA. awamori are greatly enhanced if prochymosin is synthesized as afusion with the carboxyl terminus of glucoamylase as compared to directexpression from the glaA promoter utilizing the glucoamylase signalpeptide. Increased efficiency of secretion of the fusion proteincompared to prochymosin appears to be at least part of the explanationfor higher expression levels. This is apparent from the high proportionof chymosin found within the cell in pGRG1 and pGRG3 transformantscompared to pGAMpR transformants. Neither authentic bovine chymosin, northe majority of the chymosin produced in A. awamori are glycosylated.Attachment of prochymosin to glucoamylase, which is extensivelydecorated with O-linked carbohydrates in A. niger (Pazur, J. H. et al.(1987) J. Protein Chem. 6, 517-527), may allow more efficient passagethrough the Aspergillus secretory pathway.

The plasmids pGRG1 and pGRG3 both employ an A. niger glaA promoter todirect chymosin expression, whereas an A. awamori glaA promoter ispresent in pGAMpR. There are additional differences between theseplasmids such as the inclusion of the ans1 sequence on pGRG1 and pGRG3.Integration of the various plasmids was probably not by homology withthe native glaA locus. Consequently, the chromosomal location of theintegrated plasmids was presumably different in each transformant. Allof these differences make it difficult to compare the total amounts ofchymosin produced (intracellular plus extracellular) and to determine ifthis is similar between transformants, with the distribution between theinside and the outside of the cell being the only distinction betweendirect expression and production as a fusion protein. Northern analysissuggested that the steady state level of chymosin-specific mRNA werehigher in transformant 12grg1-1a than in 12gampr4, making somecomparison of chymosin yields valid.

Analysis of the total amount of chymosin produced by transformants12grg1-1a, 12gampr4 and 23gampr46 in soy medium showed that the totalamount of chymosin produced in the direct expression transformant wasmuch less than that in the transformants expressing chymosin as a fusionprotein. This might suggest that enhanced efficiency of secretion maynot be the only benefit of expression of chymosin fused to glucoamylase.It is possible that translation of the glucoamylase-chymosin fusion mRNAwas more efficient than translation of prochymosin mRNA in which onlythe untranslated leader sequence and the secretion signal sequence wasderived from the glaA gene. However, it may be difficult to get anaccurate value for intracellular chymosin levels since extraction maynot be complete and the NaOH treatment required to release chymosinreduced detection by EIA. Additionally, chymosin which accumulatesintracellularly may be subject to degradation by native proteases priorto extraction. Consequently, the figures obtained for intracellularchymosin concentrations will always be underestimates.

It was apparent, using samples from young cultures in soy bean mealmedium at pH 6, that a large proportion of the glucoamylase-prochymosinfusion protein was secreted to the medium intact, although some maturechymosin was also observed. Under these conditions prochymosin was theonly form detected by Western analysis of samples from the directexpression transformant 12grg1-1a. In contrast, only mature chymosin wasdetected in cultures of any of the transformants in SCM at pH 5. Theseobservations suggested that the release of chymosin from theglucoamylase-chymosin fusion protein was favored at low pH and mayinvolve the natural autocatalytic activation mechanism of prochymosin.Loss of the fusion protein and an increase in active chymosinconcentration could be induced simply by lowering the pH of samples to2. As might be expected if processing was dependent on chymosinactivity, at least some of the chymosin released from the fusion proteinunder these conditions appeared to be in the form of pseudochymosin.Presumably, this would eventually be further processed to maturechymosin under the appropriate conditions. Processing of the fusionprotein at pH 2 was inhibited by pepstatin suggesting that it requiredthe activity of an aspartyl protease. This activity could be supplied bychymosin itself or by a native A. awamori protease. We have constructeda strain of A. awamori in which the gene encoding the major secretedaspartyl protease, aspergillopepsin A, has been deleted (see U.S. patentapplication Ser. No. 214,237, abandoned). Although there is a low levelof extracellular proteolytic activity remaining in this strain, thisactivity is unaffected by pepstatin. Processing of theglucoamylase-chymosin fusion in this aspergillopepsin-deleted strain isindistinguishable from the processing described above for transformants12gampr4 and 23gampr46 (data not shown). This is further indication thatthe pepstatin-inhibitable activity which causes processing of the fusionprotein is actually that of chymosin itself.

Amino terminal sequencing of the mature chymosin obtained from pGAMpRtransformants has confirmed that correct processing has occurred(results not shown). Other tests, including amino acid compositionanalysis, specific activity determination, Ouchterlony plate tests andcheese-making trials, have confirmed the authenticity of the chymosinproduced in these transformants.

EXAMPLE 2 Secretion of Chymosin from a Fusion Polypeptide Containing A.awamori α-amylase

Construction of pAMpRI and pAMpRII

Aspergillus awamori strain UVK143f has two almost identical α-amylasegenes (amyA and amyB), both of which had been cloned and one of whichhas been sequenced (Korman, D. R., (1988). The cloning andcharacterization of α-amylase genes of Aspergillus oryzae andAspergillus awamori. MA thesis, San Fransisco State University).Subsequently, the second gene has been sequenced. The amyA gene encodesa protein of 496 amino acids including an amino terminal signal sequenceof 21 amino acids. The sequence for each of the two genes is identical,including 200 bp of sequence 5' to the translation start codon, theposition and sequence of the eight introns and the entire codingsequence except for the sequence encoding the final two or threecarboxyl terminal amino acids (codons for tyrosine and glycine in amyAare replaced by three serine codons in amyB). The vectors pUCAMpRI andpUCAMpRII contain similar prochymosin B expression cassettes to pGAMpRexcept that the promoter, the entire coding sequence of the A. awamoriamyA or amyB genes and the amyA terminator and polyadenylation sequencereplace those of the glaA gene.

Construction of pUCAMpRI is described in FIG. 6. Briefly, a syntheticoligonucleotide encoding the last five amino acids of α-amylase (amyAversion) and the first six amino acids of prochymosin was used to linkexactly and in frame a BamHI-Asp718 fragment encoding a region of theprochymosin B coding sequence starting at the seventh codon with aBglI-HindIII fragment encoding the promoter region (up to 617 bp 5' ofthe translation start codon) and all of the coding sequence of amyA upto the sixth codon prior to the translation stop codon (pUCAMYint. #2).The remaining portion of the chymosin coding sequence was added as anAsp718-XbaI fragment taken from a GRG1 type expression cassette to givepUCAMYint. #3 (i.e., the XbaI site 11 bp after the translation stopcodon being an engineered site introduced during construction of pGRG1).Site directed mutagenesis was used to introduce an XbaI site 11 bp afterthe translation stop codon of the amyA gene so that the terminator andpolyadenylation region (581 bp) from this gene could be placedimmediately after the prochymosin sequence in pUCAMYint. #3 to givepUCAMpR1. The vector pUCAMpRII was essentially the same except that theamyA promoter was exchanged for the corresponding region of the amyBgene.

Secretion of Chymosin

The plasmids pUCAMpRI and pUCAMpRII contain no gene which could be usedas a selectable marker for transformation into filamentous fungi. It wastherefore necessary to introduce these plasmids into A. awamori bycotransformation with a second plasmid which did contain a selectablemarker. Consequently approximately 10 μg of pUCAMpRI or pUCAMpRII wasmixed with approximately 2 μg of pBH2 (pUC18 with a 2.4 kb BamHI-HindIIIfragment containing the Asperaillus niger pyrG gene) and used totransform strain ΔAP3 (described in U.S. patent application Ser. No.214,237, abandoned) using complementation of the pyrG mutation in thisstrain as a selection system for transformants. A proportion of thetransformants obtained in this manner should contain both plasmids.Individual transformants were subsequently grown in 1 ml liquid culturesin 24 well microtiter plates and the culture supernatants assayed forchymosin activity. Those transformants which produced the greatestamount of active, secreted chymosin were then grown in 50 ml shake flaskcultures in soy bean meal medium for further analysis. The greatestamount of chymosin produced by transformants of strain ΔAP3 was similarregardless of whether pUCAMpRI or pUCAMpRII was used for transformation.The maximum production observed was 70-80 μg/ml of chymosin. This ishigher than the level of production expected if the preprochymosin hadbeen fused directly to the α-amylase promoter without inclusion of theα-amylase coding sequence. Western immunoblotting analysis of culturesupernatants using anti-chymosin antibodies for specific staining showedthat, as with pGAMpR transformants, a fusion protein could be observedin addition to a band the size of mature chymosin (37,000 MW). Thefusion protein was of the expected size (91,000 MW) for a full lengthα-amylase/prochymosin fusion polypeptide and could also be identifiedusing anti-α-amylase antibody for specific staining. Mature chymosin wasreleased from the α-amylase/prochymosin fusion protein at low pH as wasobserved for the glucoamylase/prochymosin fusion protein.

EXAMPLE 3

Construction of pSG1

In order to test if enhanced chymosin production and secretion could beachieved by fusing a smaller part of the glucoamylase polypeptidesequence to the amino terminus of prochymosin a vector, pSG1, wasconstructed (FIG. 7). The starting point for this plasmid constructionwas a vector (pUCAagrg4) containing a XhoI/HindIII glucoamylase/chymosinexpression cassette (promoter, coding sequence and terminator regions)identical to that in pGRG4 (see EPO Publication No. 0 215 594) exceptthat the Aspergillus awamori UVK143f glaA promoter, signal sequence,prosequence and first 11 codons of the mature coding sequence replacedthe equivalent region from the A. niger glaA gene. This expressioncassette was inserted between the HindIII and SalI sites of pUC18(Yanisch-Peron, et al., 1985, Gene 33, 103-119). From pUCAagrg4 weisolated a 2.3 kb Asp718 fragment containing the A. awamori UVK143f glaApromoter, signal sequence, prosequence and first 11 codons of the matureglucoamylase coding sequence as well as an amino terminal portion of theprochymosin coding sequence. This fragment was cloned into the Asp718restriction site of pUC18 to give pUCgrg4X/K. A 1 kb BssHII fragmentfrom the coding sequence (from within the prosequence to a pointapproximately half way through the mature coding sequence) of the A.awamori glaA gene was inserted into the unique BssHII site in the glaAprosequence region of pUCgrg4X/K to give pUCgrg4X/K+B. Finally, weisolated the larger Asp718 fragment from pUCAagrg4, containing the pUCreplicon, the 3' end of the chymosin coding sequence and the A. nigerglaA polyadenylation and termination region and ligated this with thelarger Asp718 fragment from pUCgrg4X/K+B to give pSG1. Theglucoamylase/prochymosin polypeptide expected to be produced as a resultof transcription and translation should consist of glucoamylase signalsequence, glucoamylase prosequence, amino acids 1-297 of matureglucoamylase followed immediately by amino acids 1-11 of matureglucoamylase and finally prochymosin at the carboxyl terminus.

We have used pSG1 in cotransformation experiments with pBH2 but havefailed to identify transformants which produce active chymosin. Thereason for this is unclear and further work is in progress to clarifythe situation. It may be that the glucoamylase/prochymosin fusionpolypeptide encoded by this plasmid is not secreted efficiently, ormature, active chymosin may not be released from the fusion polypeptide.However, it is also conceivable that the plasmid was not constructed aspredicted, or that transcription or translation of theglucoamylase/chymosin coding sequences were not efficient.

The foregoing are presented by way of example only and should not beconstrued as a limitation to the scope of permissible claims.

Having described the preferred embodiments of the present invention, itwould appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

All references are expressly incorporated herein by reference.

What is claimed is:
 1. A fusion DNA sequence encoding a fusionpolypeptide comprising, from the 5' end of said fusion DNA sequence,first, second, third and fourth DNA sequences encoding, from the amino-to carboxy-terminus of said fusion polypeptide, corresponding first,second, third and fourth amino acid sequences, said first DNA sequenceencoding a signal peptide functional as a secretory sequence in a firstfilamentous fungus, said second DNA sequence encoding a secretedpolypeptide or portion thereof, said third DNA sequence encoding acleavable linker polypeptide and said fourth DNA sequence encoding adesired polypeptide, wherein the expression of said fusion DNA sequencein said first filamentous fungus or in a second filamentous fungusresults in increased secretion of said desired polypeptide as comparedto the secretion of said desired polypeptide from said first or saidsecond filamentous fungus when expressed as a second fusion polypeptideencoded by a second fusion DNA sequence comprising only said first,third and fourth DNA sequences.
 2. The fusion DNA sequence of claim 1wherein said first DNA sequence encodes a signal peptide selected fromthe group consisting of signal peptides from glucoamylase, α-amylase,and aspartyl protease from Aspergillus species, signal peptides frombovine chymosin and human tissue plasminogen activator and signalpeptides from Trichoderma cellobiohydrolase I and II.
 3. The fusion DNAsequence of claim 1 wherein said first DNA sequence encodes the signalpeptide from Asperaillus awamori glucoamylase.
 4. The fusion DNAsequence of claim 1 wherein said second DNA sequence encodes a secretedpolypeptide or a portion thereof selected from the group consisting ofglucoamylase, α-amylase, and aspartyl protease from Aspergillus speciesand Trichoderma cellobiohydrolase I and II.
 5. The fusion DNA sequenceof claim 1 wherein said second DNA sequence encodes glucoamylase fromAspergillus awamori or a portion thereof.
 6. The fusion DNA sequence ofclaim 1 wherein said third DNA sequence encodes a cleavable linkerpolypeptide selected from the group consisting of the prosequence fromchymosin, the prosequence of subtilisin, and sequences recognized bytrypsin factor X_(a), collagenase, clostripain, subtilisin and chymosin.7. The DNA sequence of claim 1 wherein said third DNA sequence encodesthe prosequence of chymosin or a portion thereof.
 8. The fusion DNAsequence of claim 1 wherein said fourth DNA sequence encodes a desiredpolypeptide selected from the group consisting of enzymes, proteinaceoushormones and serum proteins.
 9. The fusion DNA sequence of claim 1wherein said fourth DNA sequence encodes bovine chymosin.
 10. The fusionDNA sequence of claim 1 wherein said first DNA sequence encodes thesignal peptide from Aspergillus awamori glucoamylase, said secondsequence encodes glucoamylase from Aspergillus awamori said thirdsequence encodes the prosequence of chymosin and said fourth sequenceencodes chymosin.
 11. An expression vector for transforming a hostfilamentous fungus comprising DNA sequences encoding regulatorysequences functionally recognized by said host filamentous fungusincluding promoter and transcription and translation initiationsequences operably linked to the 5' end of the fusion DNA sequence ofclaim 1 and transcription stop sequences and polyadenylation sequencesoperably linked to the 3' end of said fusion DNA sequence.
 12. Theexpression vector of claim 11 wherein said first and said second DNAsequences encoding respectively said signal peptide and said secretedpolypeptide are selected from filamentous fungi of the same genus assaid host filamentous fungus.
 13. The expression vector of claim 12wherein said genus is selected from the group consisting of Aspergillus,Trichoderma, Neurospora, Penicillium, Cephalosporium, Podospora,Endothia, Mucor, Cochliobolus, Pyricularia, Achlya and Humicola.
 14. Theexpression vector of claim 12 wherein said genus is Aspergillus.
 15. Theexpression vector of claim 11 wherein said first and said second DNAsequences encoding respectively said signal peptide and said secretedpolypeptide are from said host filamentous fungus.
 16. A filamentousfungus comprising an expression vector, said expression vector as in oneof claims 11-15.
 17. A fusion polypeptide comprising, from the amino- tocarboxy-terminus, first, second, third and fourth amino acid sequences,said first amino acid sequence comprising a signal peptide functional asa secretory sequence in a first filamentous fungus, said second aminoacid sequence comprising a secreted polypeptide or portion thereof, saidthird amino acid sequence comprising a cleavable linker polypeptide andsaid fourth amino acid sequence comprising a desired polypeptide,wherein the expression of a fusion DNA sequence encoding said fusionpolypeptide in said first filamentous fungus or in a second filamentousfungus results in increased secretion of said desired polypeptide ascompared to the secretion of said desired polypeptide from said first orsaid second filamentous fungus when expressed from a second fusion DNAsequence encoding a second fusion polypeptide comprising only saidfirst, third and fourth amino acid sequences.
 18. The fusion polypeptideof claim 17 wherein said first amino acid sequence comprises a signalpeptide selected from the group consisting of signal peptides fromglucoamylase, α-amylase, and aspartyl protease from Aspergillus species,signal peptides from bovine chymosin and human tissue plasminogenactivator and signal peptides from Trichoderma cellobiohydrolase I andII.
 19. The fusion polypeptide of claim 17 wherein said first amino acidsequence is the signal peptide from Asperaillus awamori glucoamylase.20. The fusion polypeptide of claim 17 wherein said second amino acidsequence is selected from the group consisting of glucoamylase,α-amylase, and aspartyl protease from Aspergillus species andTrichoderma cellobiohydrolase I and II.
 21. The fusion polypeptide ofclaim 17 wherein said second amino acid sequence is glucoamylase fromAspergillus awamori.
 22. The fusion polypeptide of claim 1 wherein saidcleavable linker polypeptide is selected from the group consisting ofthe prosequence of subtilisin, and sequences recognized by trypsinfactor X_(a), collagenase, clostripain, subtilisin and chymosin.
 23. Thefusion polypeptide of claim 17 wherein said third amino acid sequence isthe prosequence of chymosin.
 24. The fusion polypeptide of claim 17wherein said fourth amino acid sequence is selected from the groupconsisting of enzymes, proteinaceous hormones and serum proteins. 25.The fusion polypeptide of claim 17 wherein said fourth amino acidsequence is chymosin.
 26. The fusion polypeptide of claim 17 whereinsaid first amino acid sequence is the signal peptide of A. awamoriglucoamylase, said third amino acid sequence is the prosequence ofchymosin and said fourth amino acid sequence is bovine chymosin.
 27. Aprocess for producing a desired polypeptide comprising:culturing a hostfilamentous fungus transformed with an expression vector comprising thefusion DNA sequence of claim 1 under conditions which permit expressionof said fusion DNA sequence to cause the secretion of the desiredpolypeptide encoded by said fusion DNA sequence.
 28. A process accordingto claim 27, wherein said first DNA sequence encodes a signal peptideselected from the group consisting of signal peptides from glucoamylase,α-amylase, and aspartyl protease from Aspergillus species, signalpeptides from bovine chymosin and human tissue plasminogen activator andsignal peptides from Trichoderma cellobiohydrolase I and II.
 29. Aprocess according to claim 27, wherein said first DNA sequence encodes asignal peptide from Aspergillus awamori glucoamylase.
 30. A processaccording to claim 27, wherein said second DNA sequence encodes asecreted polypeptide or a portion thereof selected from the groupconsisting of glucoamylase, α-amylase, and aspartyl protease fromAspergillus species and Trichoderma cellobiohydrolase I and II.
 31. Aprocess according to claim 27, wherein said second DNA sequence encodesglucoamylase from Aspergillus awamori or a portion thereof.
 32. Aprocess according to claim 27, wherein said third DNA sequence encodes acleavable linker polypeptide selected from the group consisting of theprosequence from chymosin, the prosequence of subtilisin, and sequencesrecognized by trypsin factor X_(a), collagenase, clostripain, subtilisinand chymosin.
 33. A process according to claim 27, wherein said thirdDNA sequence encodes the prosequence of chymosin or a portion thereof.34. A process according to claim 27, wherein said fourth DNA sequenceencodes a desired polypeptide selected from the group consisting ofenzymes, proteinaceous hormones and serum proteins.
 35. A processaccording to claim 27, wherein said fourth DNA sequence encodes bovinechymosin.
 36. A process according to claim 27, wherein said first DNAsequence encodes the signal peptide from Aspergillus awamoriglucoamylase, said second sequence encodes glucoamylase from Aspergillusawamori, said third sequence encodes the prosequence of chymosin andsaid fourth sequence encodes chymosin.
 37. A process according to claim27, wherein said expression vector comprises DNA sequences encodingregulatory sequences functionally recognized by said host filamentousfungus including promoter and transcription and translation initiationsequences operably linked to the 5' end of said fusion DNA sequence andtranscription stop sequences and polyadenylation sequences operablylinked to the 3' end of said fusion DNA sequence.
 38. A processaccording to claim 27, wherein said signal peptide and said secretedpolypeptide are selected from filamentous fungi of the same genus assaid host filamentous fungus.
 39. A process according to claim 38,wherein said genus is selected from the group consisting of Aspergillus,Trichoderma, Neurospora, Penicillium, Cephalosporium, Podospora,Endothia, Mucor, Cochliobolus, Pyricularia, Achlya and Humicola.
 40. Aprocess according to claim 38, wherein said genus is Aspergillus.
 41. Aprocess according to claim 38, wherein said first and said second DNAsequences encoding respectively said signal peptide and said secretedpolypeptide are from said host filamentous fungus.